This invention relates to antithrombotic aromatic amides which demonstrate activity as inhibitors of factor Xa and, accordingly, which are useful anticoagulants in mammals. In particular it relates to aromatic amides having high anticoagulant activity, and antithrombotic activity. Thus, this invention relates to new amides which are inhibitors of factor Xa, pharmaceutical compositions containing the amides as active ingredients, and the use of the amides as anticoagulants for prophylaxis and treatment of thromboembolic disorders such as venous thrombosis, pulmonary embolism, arterial thrombosis, in particular myocardial ischemia, myocardial infarction and cerebral thrombosis, general hypercoagulable states and local hypercoagulable states, such as following angioplasty and coronary bypass operations, and generalized tissue injury as it relates to the inflammatory process. In addition, the antithrombotic agents are useful as anticoagulants in in vitro applications.
The process of blood coagulation, thrombosis, is triggered by a complex proteolytic cascade leading to the formation of thrombin. Thrombin proteolytically removes activation peptides from the Axcex1-chains and the Bxcex2-chains of fibrinogen, which is soluble in blood plasma, initiating insoluble fibrin formation. The formation of thrombin from prothrombin is catalyzed by factor Xa.
Anticoagulation currently is achieved by the administration of heparins and coumarins. Parenteral pharmacological control of coagulation and thrombosis is based on inhibition of thrombin through the use of heparins. Heparins act indirectly on thrombin by accelerating the inhibitory effect of endogenous antithrombin III (the main physiological inhibitor of thrombin). Because antithrombin III levels vary in plasma and because clot-bound thrombin seems resistant to this indirect mechanism, heparins can be an ineffective treatment. Because coagulation assays are believed to be associated with efficacy and with safety, heparin levels must be monitored with coagulation assays (particularly the activated partial thromboplastin time (APTT) assay). Coumarins impede the generation of thrombin by blocking the posttranslational gamma-carboxylation in the synthesis of prothrombin and other proteins of this type. Because of their mechanism of action, the effect of coumarins can only develop slowly, 6-24 hours after administration. Further, they are not selective anticoagulants. Coumarins also require monitoring with coagulation assays (particularly the prothrombin time (PT) assay).
Recently, interest has grown in small synthetic molecules which demonstrate potent direct inhibition of thrombin and factor Xa. See, Joseph P. Vacca (Annette M. Doherty Section Editor), Annual Reports in Medicinal Chemistry, (1998), 33, 81-90.
Although the heparins and coumarins are effective anticoagulants, there still exists a need for anticoagulants which act selectively on factor Xa or thrombin, and which, independent of antithrombin III, exert inhibitory action shortly after administration, preferably by an oral route, and do not interfere with lysis of blood clots, as required to maintain hemostasis.
The present invention is directed to the discovery that the amides of the present invention, as defined below, are potent inhibitors of factor Xa which may have high bioavailability following oral administration.
According to the invention there is provided a compound of formula I 
(or a pharmaceutically acceptable salt thereof) wherein:
A3, A4, A5 and A6, together with the two carbons to which they are attached, complete a substituted benzene in which A3 is CR3, A4 is CR4, A5 is CR5, and A6 is CR6;
wherein
R3 is hydrogen, methyl, methoxy, fluoro, chloro or carboxy;
one of R4 and R5 is hydrogen, (1-4C)alkyl, halo, trifluoromethyl, trifluoromethoxy, RfOxe2x80x94, RfO2CCH2Oxe2x80x94, HO(CH2)aOxe2x80x94 (in which a is 2, 3 or 4), RfO2Cxe2x80x94, RfO2CCH2xe2x80x94, RgNHxe2x80x94, RhSO2xe2x80x94, hydroxymethyl, formyl, cyano, acetyl, 1-hydroxyethyl, 1-thydroxyimino)ethyl, 1-(methoxyimino)-ethyl, methylthio or RfO2C(CH2)2xe2x80x94;
the other of R4 and R5 is hydrogen; and
R6 is hydrogen, methyl, fluoro, chloro or methoxy;
in which Rf is hydrogen, (1-4C)alkyl or benzyl; Rg is hydrogen or RhSO2xe2x80x94; and Rh is (1-4C)alkyl or dimethylamino;
or each of R3, R4 and R6 is hydrogen; and R5 is vinyl, 2-cyanovinyl, 2-({(1-2C)alkoxy}carbonyl)vinyl or Ra in which Ra is phenyl (which is unsubstituted or bears one or more substituents independently selected from halo, methyl, methoxy and hydroxy) or heteroaryl (which heteroaryl is a 5-membered aromatic ring which includes one to four heteroatoms selected from sulfur, oxygen and nitrogen or is a 6-membered aromatic ring which includes one to three nitrogen atoms, wherein the heteroaryl is attached at carbon and may bear one or more methyl substituents on carbon or nitrogen);
L1 is xe2x80x94COxe2x80x94NHxe2x80x94 such that xe2x80x94L1xe2x80x94Q1 is xe2x80x94COxe2x80x94NHxe2x80x94Q1;
Q1 is 2-pyridinyl (which bears a methyl, methoxy, methylthio, fluoro or chloro substituent at the 5-position), 3-pyridinyl (which bears a methyl, fluoro or chloro substituent at the 6-position), 2-pyrimidinyl (which may bear a methyl, fluoro or chloro substituent at the 5-position) or 3-pyridazinyl (which may bear a methyl, fluoro or chloro substituent at the 6-position);
R2 is xe2x80x94L2xe2x80x94Q2 in which xe2x80x94L2xe2x80x94 is xe2x80x94NHxe2x80x94COxe2x80x94, xe2x80x94NHxe2x80x94COxe2x80x94Xxe2x80x94, xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94Xxe2x80x94, xe2x80x94NHxe2x80x94COxe2x80x94NHxe2x80x94Xxe2x80x94, xe2x80x94NHxe2x80x94CH2xe2x80x94, xe2x80x94NHxe2x80x94C(CH3)Hxe2x80x94, xe2x80x94N(CH3)xe2x80x94CH2xe2x80x94 or xe2x80x94Oxe2x80x94CH2xe2x80x94; and Q2 is Q2A, Q2B, Q2C, Q2D, Q2E or Q2F wherein X is a single bond or methylene and the values of L2 and Q2 are together selected from xe2x80x94NHxe2x80x94COxe2x80x94Xxe2x80x94Q2A, xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94Xxe2x80x94Q2A, xe2x80x94NHxe2x80x94COxe2x80x94NHxe2x80x94Xxe2x80x94Q2A, xe2x80x94NHxe2x80x94CH2xe2x80x94Q2A, xe2x80x94NHxe2x80x94C(CH3)Hxe2x80x94Q2A, xe2x80x94N(CH3)xe2x80x94CH2xe2x80x94Q2A, xe2x80x94Oxe2x80x94CH2xe2x80x94Q2A, xe2x80x94NHxe2x80x94COxe2x80x94Xxe2x80x94Q2B, xe2x80x94NHxe2x80x94COxe2x80x94Q2C, xe2x80x94NHxe2x80x94COxe2x80x94Q2D, xe2x80x94NHxe2x80x94COxe2x80x94Q2E and xe2x80x94NHxe2x80x94COxe2x80x94Q2F in which:
Q2A (showing the L2 to which it is attached) is 
in which
each of m and n independently is 0 or 1, or m is 2 and n is 1, and
R2A is hydrogen, t-butyl, methylsulfonyl, xe2x80x94CHRyRz, xe2x80x94CHRwRx, or 4-pyridinyl (which is unsubstituted or bears a substituent Rv at the 2- or 3-position) where in
Rv is methyl, hydroxymethyl, {(1-2C) alkoxy}carbonyl; cyano, carbamoyl, thiocarbamoyl, or N-hydroxyamidino;
each of Rw and Rx independently is hydrogen or (1-3C)normal alkyl; or xe2x80x94CHRwRx is 2-indanyl or (showing the nitrogen to which it is attached) is 
in which T is a single bond or methylene and U is methylene, ethylene, oxy, xe2x80x94S(O)qxe2x80x94 (wherein q is 0, 1 or 2) or imino (which may bear a methyl substituent), or T is ethan-1,1-diyl and U is a single bond or methylene;
Ry is hydrogen or methyl; and
Rz is isopropyl, t-butyl, (3-6C)cycloalkyl, phenyl (which is unsubstituted or bears one or more substituents independently selected from halo, methyl, methoxy and hydroxy), 4-quinolinyl or heteroaryl (which heteroaryl is a 5-membered aromatic ring which includes one to four heteroatoms selected from sulfur, oxygen and nitrogen or is a 6-membered aromatic ring which includes one to three nitrogen atoms, wherein the heteroaryl is attached at carbon and may bear one or more methyl substituents on carbon or nitrogen);
or R2A is xe2x80x94Lbxe2x80x94CH2xe2x80x94Rb in which xe2x80x94Lbxe2x80x94 is a direct bond, xe2x80x94CH2xe2x80x94, xe2x80x94C(CH3)Hxe2x80x94 or xe2x80x94CH2xe2x80x94CH2xe2x80x94; and Rb is carboxy, {(1-2C)alkoxy}carbonyl, cyano, carbamoyl or trifluoromethyl;
or R2A is xe2x80x94COxe2x80x94Rc in which Rc is hydrogen, (1-3C)alkyl, {(1-2C)alkoxy}carbonyl-(CH2)cxe2x80x94 (in which c is 1 or 2), phenyl (which is unsubstituted or bears one or more substituents independently selected from halo, methyl, methoxy and hydroxy), heteroaryl (which heteroaryl is a 5-membered aromatic ring which includes one to four heteroatoms selected from sulfur, oxygen and nitrogen or is a 6-membered aromatic ring which includes one to three nitrogen atoms, wherein the heteroaryl is attached at carbon and may bear one or more methyl substituents on carbon or nitrogen) or xe2x80x94NRdRe in which each of Rd and Re is independently hydrogen, methyl or ethyl, or xe2x80x94NRdRe is pyrrolidino, piperidino, morpholino or thiomorpholino;
Q2B is 1-piperazinyl which bears at the 4-position the group R2A (defined as above);
Q2C is 3,4-didehydropiperidin-4-yl which bears at the 1-position the group R2A (defined as above);
Q2D is cyclohexyl which bears at the 4-position the group xe2x80x94NRsRt in which each of Rs and Rt independently is hydrogen or methyl or Rs and Rt together are trimethylene or tetramethylene;
Q2E is 1-piperidinyl which bears at the 4-position the group xe2x80x94NRsRt (defined as above); and
Q2F (showing the L2 to which it is attached) is 
in which Ro is hydrogen, halo, (1-6C)alkyl, hydroxy, (1-4C)alkoxy, benzyloxy or (1-4C)alkylthio; and Rp is acetylamino, 1-hydroxyethyl, 1-hydroxy-1-methylethyl, 1-methoxy-1-methylethyl, 4-piperidinyl, 4-pyridinyl, dimethylaminosulfonyl or xe2x80x94Jxe2x80x94Rq in which J is a single bond, methylene, carbonyl, oxy, xe2x80x94S(O)qxe2x80x94 (wherein q is 0, 1 or 2), or xe2x80x94NRrxe2x80x94 (wherein Rr is hydrogen or methyl); and Rq is (1-6C)alkyl, phenyl, 3-pyridyl or 4-pyridyl; or xe2x80x94NRqRr is pyrrolidino.
A particular compound of formula I is one wherein:
A3, A4, A5 and A6, together with the two carbons to which they are attached, complete a substituted benzene in which A3 is CR3, A4 is CR4, A5 is CR5, and A6 is CR6;
wherein
R3 is hydrogen, methyl, fluoro, chloro or carboxy;
one of R4 and R5 is hydrogen, (1-4C)alkyl, halo, trifluoromethyl, trifluoromethoxy, RfOxe2x80x94, RfO2CCH2Oxe2x80x94, HO(CH2)aOxe2x80x94 (in which a is 2, 3 or 4), RfO2Cxe2x80x94, RfO2CCH2xe2x80x94, RgNHxe2x80x94 or RhSO2xe2x80x94;
the other of R4 and R5 is hydrogen; and
R6 is hydrogen, methyl, fluoro, chloro or methoxy;
in which Rf is hydrogen, (1-4C)alkyl or benzyl; Rg is hydrogen or RhSO2xe2x80x94; and Rh is (1-4C)alkyl or dimethylamino;
L1 is xe2x80x94COxe2x80x94NHxe2x80x94 such that xe2x80x94L1xe2x80x94Q1 is xe2x80x94COxe2x80x94NHxe2x80x94Q1;
Q1 is 2-pyridinyl (which bears a methyl, methoxy, methylthio, fluoro or chloro substituent at the 5-position), 3-pyridinyl (which bears a methyl, fluoro or chloro substituent at the 6-position), 2-pyrimidinyl (which may bear a methyl, fluoro or chloro substituent at the 5-position) or 3-pyridazinyl (which may bear a methyl, fluoro or chloro substituent at the 6-position);
R2 is xe2x80x94L2xe2x80x94Q2 in which xe2x80x94L2xe2x80x94 is xe2x80x94NHxe2x80x94COxe2x80x94, xe2x80x94NHxe2x80x94COxe2x80x94Xxe2x80x94, xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94Xxe2x80x94, xe2x80x94NHxe2x80x94COxe2x80x94NHxe2x80x94Xxe2x80x94, xe2x80x94NHxe2x80x94CH2xe2x80x94 or xe2x80x94Oxe2x80x94CH2xe2x80x94; and Q2 is Q2A, Q2B, Q2C, Q2D, Q2E or Q2F wherein X is a single bond or methylene and the values of L2 and Q2 are together selected from xe2x80x94NHxe2x80x94COxe2x80x94Xxe2x80x94Q2A, xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94Xxe2x80x94Q2A, xe2x80x94NHxe2x80x94COxe2x80x94NHxe2x80x94Xxe2x80x94Q2A, xe2x80x94NHxe2x80x94CH2Q2A, xe2x80x94Oxe2x80x94CH2Q2A, xe2x80x94NHxe2x80x94COxe2x80x94Xxe2x80x94Q2B, xe2x80x94NHxe2x80x94COxe2x80x94Q2C, xe2x80x94NHxe2x80x94COxe2x80x94Q2D, xe2x80x94NHxe2x80x94COxe2x80x94Q2E and xe2x80x94NHxe2x80x94COxe2x80x94Q2F in which:
Q2A (showing the L2 to which it is attached) is 
in which
each of m and n independently is 0 or 1, and
R2A is hydrogen, t-butyl, methylsulfonyl, xe2x80x94CHRyRz, xe2x80x94CHRwRx, or 4-pyridinyl (which is unsubstituted or bears a substituent Rv at the 2- or 3-position) wherein
Rv is methyl, hydroxymethyl, ((1-2C)alkoxy)carbonyl; cyano, carbamoyl, thiocarbamoyl, or N-hydroxyamidino;
each of Rw and Rx independently is hydrogen or (1-3C)normal alkyl; or xe2x80x94CHRwRx is 2-indanyl or (showing the nitrogen to which it is attached) is 
in which T is a single bond or methylene and U is methylene, ethylene, oxy, xe2x80x94S(O)qxe2x80x94 (wherein q is 0, 1 or 2) or imino (which may bear a methyl substituent), or T is ethan-1,1-diyl and U is a single bond or methylene;
Ry is hydrogen or methyl; and
Rz is isopropyl, t-butyl, (3-6C)cycloalkyl, phenyl (which is unsubstituted or bears one or more substituents independently selected from halo, methyl, methoxy and hydroxy), 4-quinolinyl or heteroaryl (which heteroaryl is a 5-membered aromatic ring which includes one to four heteroatoms selected from sulfur, oxygen and nitrogen or is a 6-membered aromatic ring which includes one to three nitrogen atoms, wherein the heteroaryl is attached at carbon and may bear one or more methyl substituents on carbon or nitrogen);
Q2B is 1-piperazinyl which bears at the 4-position the group R2A (defined as above);
Q2C is 3,4-didehydropiperidin-4-yl which bears at the 1-position the group R2A (defined as above);
Q2D is cyclohexyl which bears at the 4-position the group xe2x80x94NRsRt in which each of Rs and Rt independently is hydrogen or methyl or Rs and Rt together are trimethylene or tetramethylene;
Q2E is 1-piperidinyl which bears at the 4-position the group xe2x80x94NRsRt (defined as above); and
Q2F (showing the L2 to which it is attached) is 
in which Ro is hydrogen, halo, (1-6C)alkyl, hydroxy, (1-4C)alkoxy, benzyloxy or (1-4C)alkylthio; and Rp is acetylamino, 1-hydroxyethyl, 1-hydroxy-1-methylethyl, 1-methoxy-1-methylethyl, 4-piperidinyl, 4-pyridinyl, dimethylaminosulfonyl or xe2x80x94Jxe2x80x94Rq in which J is a single bond, methylene, carbonyl, oxy, xe2x80x94S(O)qxe2x80x94 (wherein q is 0, 1 or 2), or xe2x80x94NRrxe2x80x94 (wherein Rr is hydrogen or methyl); and Rq is (1-6C)alkyl, phenyl, 3-pyridyl or 4-pyridyl.
As used herein, the expression a compound of formula I or the expression a compound of the invention includes the compound and any conventional prodrug thereof, as well as a pharmaceutically acceptable salt of said compound or prodrug.
A more particular compound of formula I is one wherein
A3, A4, A5 and A6, together with the two carbons to which they are attached, complete a substituted benzene in which A3 is CR3, A4 is CR4, A5 is CR5, and A6 is CR6;
wherein
R3 is hydrogen;
one of R4 and R5 is hydrogen, methyl, fluoro, chloro, trifluoromethyl, trifluoromethoxy, RfO2Cxe2x80x94 or RgNHxe2x80x94;
the other of R4 and R5 is hydrogen; and
R6 is hydrogen;
in which Rf is hydrogen, (1-4C)alkyl or benzyl; Rg is hydrogen or RhSO2xe2x80x94; and Rh is (1-4C)alkyl or dimethylamino;
L1 is xe2x80x94COxe2x80x94NHxe2x80x94 such that xe2x80x94L1xe2x80x94Q1 is xe2x80x94COxe2x80x94NHxe2x80x94Q1;
Q1 is 2-pyridinyl (which bears a methyl, fluoro or chloro substituent at the 5-position), 3-pyridinyl (which bears a methyl, fluoro or chloro substituent at the 6-position), 2-pyrimidinyl (which may bear a methyl, fluoro or chloro substituent at the 5-position) or 3-pyridazinyl (which may bear a methyl, fluoro or chloro substituent at the 6-position);
R2 is xe2x80x94L2xe2x80x94Q2 in which xe2x80x94L2xe2x80x94 is xe2x80x94NHxe2x80x94COxe2x80x94, xe2x80x94NHxe2x80x94COxe2x80x94Xxe2x80x94, xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94Xxe2x80x94, xe2x80x94NHxe2x80x94COxe2x80x94NHxe2x80x94Xxe2x80x94, xe2x80x94NHxe2x80x94CH2xe2x80x94 or xe2x80x94Oxe2x80x94CH2xe2x80x94; and Q2 is Q2A, Q2B, Q2C, Q2D, Q2E or Q2F wherein X is a single bond or methylene and the values of L2 and Q2 are together selected from xe2x80x94NHxe2x80x94COxe2x80x94Xxe2x80x94Q2A, xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94Xxe2x80x94Q2A, xe2x80x94NHxe2x80x94COxe2x80x94NHxe2x80x94Xxe2x80x94Q2A, xe2x80x94NHxe2x80x94CH2xe2x80x94Q2A, xe2x80x94Oxe2x80x94CH2xe2x80x94Q2A, xe2x80x94NHxe2x80x94COxe2x80x94Xxe2x80x94Q2B, xe2x80x94NHxe2x80x94COxe2x80x94Q2C, xe2x80x94NHxe2x80x94COxe2x80x94Q2D, xe2x80x94NHxe2x80x94COxe2x80x94Q2E and xe2x80x94NHxe2x80x94COxe2x80x94Q2F in which:
Q2A (showing the L2 to which it is attached) is 
in which
each of m and n independently is 0 or 1, and
R2A is hydrogen, xe2x80x94CHRyRz, xe2x80x94CHRwRx, or 4-pyridinyl (which is unsubstituted or bears a substituent Rv at the 2- or 3-position) wherein
Rv is methyl, hydroxymethyl, {(1-2C) alkoxy}carbonyl; cyano, carbamoyl, thiocarbamoyl, or N-hydroxyamidino;
each of Rw and Rx independently is hydrogen or (1-3C)normal alkyl; or xe2x80x94CHRwRx is 2-indanyl or (showing the nitrogen to which it is attached) is 
in which T is a single bond or methylene and U is methylene, oxy, thioxy or imino (which may bear a methyl substituent), or T is ethan1,1-diyl and U is a single bond or methylene;
Ry is hydrogen or methyl; and
Rz is isopropyl, t-butyl, (3-6C)cycloalkyl, phenyl (which is unsubstituted or bears one or more substituents independently selected from halo, methyl, methoxy and hydroxy), 4-quinolinyl or heteroaryl (which heteroaryl is a 5-membered aromatic ring which includes one to four heteroatoms selected from sulfur, oxygen and nitrogen or is a 6-membered aromatic ring which includes one to three nitrogen atoms, wherein the heteroaryl is attached at carbon and may bear one or more methyl substituents on carbon or nitrogen);
Q2B is 1-piperazinyl which bears at the 4-position the group R2A (defined as above);
Q2C is 3,4-didehydropiperidin-4-yl which bears at the 1-position the group R2A (defined as above);
Q2D is cyclohexyl which bears at the 4-position the group xe2x80x94NRsRt in which each of Rs and Rt independently is hydrogen or methyl or Rs and Rt together are trimethylene or tetramethylene;
Q2E is 1-piperidinyl which bears at the 4-position the group xe2x80x94NRsRt (defined as above); and
Q2F (showing the L2 to which it is attached) is 
in which Ro is hydrogen and Rp is acetylamino, 1-hydroxyethyl, 1-hydroxy-1-methylethyl, 1-methoxy-1-methylethyl, 4-piperidinyl, 4-pyridinyl, dimethylaminosulfonyl or xe2x80x94Jxe2x80x94Rq in which J is a single bond, methylene, carbonyl, oxy, xe2x80x94S(O)qxe2x80x94 (wherein q is 0, 1 or 2), or xe2x80x94NRrxe2x80x94 (wherein Rr is hydrogen or methyl); and Rq is (1-6C)alkyl, phenyl, 3-pyridyl or 4-pyridyl.
A pharmaceutically acceptable salt of an antithrombotic agent of the instant invention includes one which is an acid-addition salt made from a basic compound of formula I and an acid which provides a pharmaceutically acceptable anion, as well as a salt which is made from an acidic compound of formula I and a base which provides a pharmaceutically acceptable cation. Thus, a salt of a novel compound of formula I as provided herein made with an acid or base which affords a pharmaceutically acceptable counterion provides a particular aspect of the invention. Examples of such acids and bases are provided hereinbelow.
As an additional aspect of the invention there is provided a pharmaceutical formulation comprising in association with a pharmaceutically acceptable carrier, diluent or excipient, a novel compound of formula I (or a pharmaceutically acceptable salt thereof) as provided in any of the descriptions herein.
In addition, there is provided the use of a factor Xa inhibiting compound of formula I (or prodrug or salt) as described herein as an active ingredient in the manufacture of a medicament for use in producing an anticoagulant or antithrombotic effect.
The present invention also provides a method of inhibiting coagulation in a mammal comprising administering to a mammal in need of treatment, a coagulation inhibiting dose of a factor Xa inhibiting compound of formula I having any of the definitions herein.
The present invention further provides a method of inhibiting factor Xa comprising administering to a mammal in need of treatment, a factor Xa inhibiting dose of a factor Xa inhibiting compound of formula I having any of the definitions herein.
Further, the present invention provides a method of treating a thromboembolic disorder comprising administering to a mammal in need of treatment, an effective dose of a factor Xa inhibiting compound of formula I having any of the definitions herein.
In addition, there is provided the use of a factor Xa inhibiting compound of formula I having any of the definitions herein for the manufacture of a medicament for treatment of a thromboembolic disorder.
As an additional feature of the invention there is provided a pharmaceutical formulation comprising in association with a pharmaceutically acceptable carrier, diluent or excipient, a prodrug of a factor Xa inhibiting compound of formula I (or of a pharmaceutically acceptable salt thereof) as provided in any of the descriptions herein.
In this specification, the following definitions are used, unless otherwise described: Halo is fluoro, chloro, bromo or iodo. Alkyl, alkoxy, etc. denote both straight and branched groups; but reference to an individual radical such as xe2x80x9cpropylxe2x80x9d embraces only the straight chain (xe2x80x9cnormalxe2x80x9d) radical, a branched chain isomer such as xe2x80x9cisopropylxe2x80x9d being specifically denoted.
Particular values are listed below for radicals, substituents, and ranges, for illustration only, and they do not exclude other defined values or other values within defined ranges for the radicals and substituents.
For an alkyl group or the alkyl portion of an alkyl containing group such as, for example alkoxy, a particular value for (1-2C)alkyl is methyl or ethyl, and more particularly is methyl; for (1-3C)normal alkyl is methyl, ethyl or propyl; for (1-4C)alkyl is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or t-butyl, and more particularly is methyl, isopropyl, butyl or t-butyl; for (1-6C)alkyl is methyl, ethyl, propyl, butyl, pentyl or hexyl, and more particularly is methyl, butyl, or hexyl. A particular value for (3-6C)cycloalkyl is cyclopropyl, cyclobutyl, cyclopenytyl or cyclohexyl. A particular value for halo is bromo or chloro, and more particularly is chloro.
A particular value for Q1 is 5-chloropyridin-2-yl, 5-fluoropyridin-2-yl, or 6-chloropyridazin-3-yl. A particular value for R2 is (1-isopropylpiperidin-4-ylcarbonyl)amino, (1-cyclohexylpiperidin-4-ylcarbonyl)amino, (4-isopropylpiperazin-1-ylcarbonyl)amino, [1-(tetrahydro-pyran-4-yl)piperidin-4-ylcarbonyl]amino, [4-(1-pyrrolidinyl)piperidin-1-ylcarbonyl]amino, [1-(4-pyridinyl)piperidin-4-ylmethyl]amino, [1-(2-carboxypyridin-4-yl)piperidin-4-ylmethyl]amino, or [1-(2-methoxycarbonylpyridin-4-yl)-piperidin-4-ylmethyl]amino. A particular set of values for R3-R6 is that each of R3-R6 is hydrogen. Another particular set of values for R3-R6 is that each of R3, R4 and R6 is hydrogen and R5 is chloro or fluoro. A further particular set of values for R3-R6 is that each of R3, R4 and R6 is hydrogen and R5 is Ra wherein Ra is phenyl, furanyl, thienyl, 2-isothiazolyl or pyridyl.
A particular species is one those listed below as example 44, 51, 69, 70, 75, 80, 84, 96 or 97.
It will be appreciated that certain compounds of formula I (or salts or prodrugs, etc.) may exist in, and be isolated in, isomeric forms, including tautomeric forms, cis- or trans-isomers, as well as optically active, racemic, or diastereomeric forms. It is to be understood that the present invention encompasses a compound of formula I in any of the tautomeric forms or as an a mixture thereof; or as a mixture of diastereomers, as well as in the form of an individual diastereomer, and that the present invention encompasses a compound of formula I as a mixture of enantiomers, as well as in the form of an individual enantiomer, any of which mixtures or form possesses inhibitory properties against factor Xa, it being well known in the art how to prepare or isolate particular forms and how to determine inhibitory properties against factor Xa by standard tests including those described below.
In addition, a compound of formula I (or salt or prodrug, etc.) may exhibit polymorphism or may form a solvate with water or an organic solvent. The present invention also encompasses any such polymorphic form, any solvate or any mixture thereof.
A prodrug of a compound of formula I may be one formed in a conventional manner with a functional group of the compound, such as with an amino, hydroxy or carboxy group.
A compound of formula I may be prepared by processes which include processes known in the chemical art for the production of structurally analogous compounds or by a novel process described herein. A process for the preparation of a compound of formula I (or a pharmaceutically acceptable salt thereof) and novel intermediates for the manufacture of a compound of formula I as defined above provide further features of the invention and are illustrated by the following procedures in which the meanings of the generic radicals are as defined above, unless otherwise specified. It will be recognized that it may be preferred or necessary to prepare a compound of formula I in which a functional group is protected using a conventional protecting group, then to remove the protecting group to provide the compound of formula I.
Thus, there is provided a process for preparing a compound of formula I (or a pharmaceutically acceptable salt thereof) as provided in any of the above descriptions which is selected from any of those described in the examples, including the following.
(A) For a compound of formula I in which xe2x80x94L2xe2x80x94Q2, is xe2x80x94NHxe2x80x94COxe2x80x94Q2, xe2x80x94NHxe2x80x94COxe2x80x94Xxe2x80x94Q2, xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94Xxe2x80x94Q2 or xe2x80x94NHxe2x80x94COxe2x80x94NHxe2x80x94Xxe2x80x94Q2, acylating an amine of formula II, 
using a corresponding acid of formula HOxe2x80x94COxe2x80x94Q2, HOxe2x80x94COxe2x80x94Xxe2x80x94Q2, HOxe2x80x94COxe2x80x94Oxe2x80x94Xxe2x80x94Q2, or HOxe2x80x94COxe2x80x94NHxe2x80x94Xxe2x80x94Q2, or an activated derivative thereof. Typical activated derivatives include the acid halides, activated esters, including 4-nitrophenyl esters and those derived from coupling reagents, as well as (when the product is a urea) isocyanates. Typical procedures include those described at example 1-D, example 4-B and example 9-A.
(B) For a compound of formula I in which xe2x80x94L2xe2x80x94Q2 is xe2x80x94Oxe2x80x94CH2xe2x80x94Q2A, akylating a phenol of formula III 
using a reagent of formula Yxe2x80x94CH2xe2x80x94Q2A in which Y is a conventional leaving group. As used herein, a leaving group xe2x80x9cYxe2x80x9d is a moiety which is displaced in a nucleophilic substitution reaction, for example a halo group (such as chloro, bromo or iodo), a sulfonate ester group (such as methylsulfonyloxy, p-toluylsulfonyloxy or trifluoromethyl-sulfonyloxy), or the reactive species derived from treating an alcohol with triphenylphospine, diethyl azodicarboxylate and triethyl amine (in a Mitsunobu reaction), for example as described at example 8-B.
(C) Acylating an amine of formula H2Nxe2x80x94Q1, or a deprotonated derivative thereof, using an acid of formula IV, or an activated derivative thereof. 
Typical deprotonated derivatives of the amine H2Nxe2x80x94Q1 include, for example, that derived from treatment of the amine with an organomagnesium reagent, for example, with allylmagnesium bromide or methylmagnesium bromide. Typical activated derivatives include the acid halides, activated esters, including 4-nitrophenyl esters and those derived from coupling reagents.
For a compound of formula I in which R2 is of the form xe2x80x94NHxe2x80x94COxe2x80x94Q2, the activated acid may be a [1,3]oxazine of formula IVa, 
wherein Q2 represents, for example, Q2A, Q2B, Q2C, Q2D, Q2E or Q2F. A typical procedure is one such as described at example 46-D (using potassium cyanide to promote the reaction) or at example 51-D.
For a compound of formula I in which R2 is of the form xe2x80x94NHxe2x80x94CH2xe2x80x94Q2, the activated acid may be an anhydride of formula IVb, 
wherein Q2 represents Q2A. A typical procedure is similar to that described at example 51-D for use with the activated acid of formula IVa.
(D) For a compound of formula I in which R2 is xe2x80x94NHxe2x80x94CH2xe2x80x94Q2A, alkylating an amine of formula II directly, using a compound of formula Yxe2x80x94CH2xe2x80x94Q2A, or (preferably) indirectly by reductive alkylation using an aldehyde of formula Q2Axe2x80x94CHO. In the reductive alkylation the intermediate imine of formula V or acid addition salt thereof (which provide a further aspect of the invention), 
may be formed in situ and reduced directly, or may be isolated prior to reduction, for example as described at examples 47-D and 59-B where the reduction is carried out using borane trimethylamine complex in glacial acetic acid.
(E) For a compound of formula I in which R2 is xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94Xxe2x80x94Q2A, or xe2x80x94NHxe2x80x94COxe2x80x94NHxe2x80x94Xxe2x80x94Q2A, acylating an alcohol of formula HOxe2x80x94Xxe2x80x94Q2A or an amine of formula NH2xe2x80x94Xxe2x80x94Q2A, using an activated derivative of an acid of formula VI, 
particularly, the corresponding isocyanate or 4-nitrophenyl ester. The procedure may be carried out analogously to the starting material preparation of example 51-A.
(F) For a compound of formula I in which R2 is xe2x80x94NHxe2x80x94COxe2x80x94Xxe2x80x94Q2B in which X is a single bond, acylating at the 1-position a piperazine of formula Hxe2x80x94Q2B, using an activated derivative of an acid of formula VI, particularly, the corresponding isocyanate or 4-nitrophenyl ester. The procedure may be carried out analogously to the starting material preparation of example 51-A or of example 95-A.
(G) For a compound of formula I in which R2 is xe2x80x94NHxe2x80x94COxe2x80x94Xxe2x80x94Q2B in which X is methylene, alkylating at the 1-position a piperazine of formula Hxe2x80x94Q2B, using an alkylating agent of formula VII 
in which Y is a leaving group, for example as described at example 45-B.
(H) For a compound of formula I in which R2A is methylsulfonyl, substituting the amino nitrogen of a corresponding compound of formula I in which R2A is hydrogen using an activated derivative of methanesulfonic acid, for example using methanesulfonyl chloride in the presence of added base.
(I) For a compound of formula I in which R2A is xe2x80x94CHRyRz or xe2x80x94CHRwRx, alkylating the amino nitrogen of a corresponding compound of formula I in which R2A is hydrogen using an alkylating agent of formula Yxe2x80x94CHRyRz or Yxe2x80x94CHRwRx or, preferably, reductively alkylating the amine using a compound of formula Ryxe2x80x94COxe2x80x94Rz or Rwxe2x80x94COxe2x80x94Rx. The direct alkylation may be completed in a polar solvent in the presence of a base, for example as described at example 102. The reductive alkylation conveniently is carried out, for example, using sodium cyanoborohydride in methanol/acetic acid as described at example 9-C and at examples 48 and 112 or using sodium triacetoxyborohydride in an inert solvent such as 1,2-dichloroethane along with an excess of the carbonyl compound and glacial acetic acid as described at example 27.
(J) For a compound of formula I in which R2A is 4-pyridinyl (which is unsubstituted or bears a substituent Rv at the 2- or 3-position), substituting the amino nitrogen of a corresponding compound of formula I in which R2A is hydrogen using a corresponding pyridine reagent bearing a leaving group Y at the 4-position, for example with a 4-chloropyridine in ethanol as described at example 52 or at example 85.
(K) For a compound of formula I in which R2A is 4-pyridinyl in which Rv is alkoxycarbonyl, esterifying a corresponding compound of formula I in which Rv is carboxy, for example as described at example 97.
(L) For a compound of formula I in which R2A is 4-pyridinyl in which Rv is hydroxymethyl, reducing the ester of a corresponding compound of formula I in which Rv is alkoxycarbonyl.
(M) For a compound of formula I in which R2A is 4-pyridinyl in which Rv is carbamoyl, amidating the ester of a corresponding compound of formula I in which Rv is alkoxycarbonyl.
(N) For a compound of formula I in which R2A is 4-pyridinyl in which Rv is thiocarbamoyl, adding H2S to the nitrile of a corresponding compound of formula I in which Rv is cyano.
(O) For a compound of formula I in which R2A is 4-pyridinyl in which Rv is N-hydroxyamidino, adding H2NOH to the nitrile of a corresponding compound of formula I in which Rv is cyano. The addition may be direct or indirect, such as via an imidate ester or by treating a compound in which Rv is thiocarbamoyl with methyl iodide to form a thioimidate ester, then treatment with hydroxylamine.
(P) For a compound of formula I in which R2A is 4-pyridinyl in which Rv is carboxy, decomposing the ester of a corresponding compound of formula I in which Rv is alkoxycarbonyl.
(Q) For a compound of formula I in which xe2x80x94NRsRt is other than amino, alkylating a corresponding compound of formula I in which xe2x80x94NRsRt is amino using a conventional method. When Rs and Rt together are trimethylene or tetramethylene, a difunctional alkylating agent, such as 1,3-dibromopropane or 1,4-dibromobutane is preferred.
(R) For a compound of formula I which bears xe2x80x94NRsRt, reductively alkylating Hxe2x80x94NRsRt using a corresponding compound but in which the carbon to bear the xe2x80x94NRsRt group bears an oxo group, for example, using a procedure similar to one of procedure (I) above, as described at example 98.
(S) For a compound of formula I in which Rp is 1-hydroxy-1-methylethyl, adding a methyl group to the carbonyl group of a corresponding compound of formula I in which Rp is acetyl using an organometallic reagent such as, for example, methylmagnesium bromide.
(T) For a compound of formula I in which Rp is 1-methoxy-1-methylethyl, treating a corresponding compound of formula I in which Rp is 1-hydroxy-1-methylethyl with methanol and an acid catalyst.
(U) For a compound of formula I in which R4 or R5 is amino, reducing the nitro group of a compound corresponding to a compound of formula I but in which R4 or R5 is nitro.
(V) For a compound of formula I in which R4 or R5 is RgNHxe2x80x94 and Rg is RhSO2xe2x80x94, substituting the amino group of a corresponding compound of formula I in which R4 or R5 is amino using an activated derivative of the sulfonic acid RhSO2xe2x80x94OH.
Whereafter, for any of the above procedures, when a functional group is protected using a protecting group, removing the protecting group.
Whereafter, for any of the above procedures, when a pharmaceutically acceptable salt of a compound of formula I is required, it is obtained by reacting the basic form of a basic compound of formula I with an acid affording a physiologically acceptable counterion or the acidic form of an acidic compound of formula I with a base affording a physiologically acceptable counterion or by any other conventional procedure.
A novel intermediate or starting material compound such as, for example, a novel compound of formula II, III, IV or VI, etc., provides a further aspect of the invention. The various starting materials may be made by processes which include processes known in the chemical art for the production of structurally analogous compounds or by a novel process described herein or one analogous thereto.
As mentioned above, a compound corresponding to a compound of formula I but in which a functional group is protected may serve as an intermediate for a compound of formula I. Accordingly, such a protected intermediate for a novel compound of formula I provides a further aspect of the invention. Thus, as one particular aspect of the invention, there is provided a compound corresponding to a novel compound of formula I as defined above in which R4 is hydroxy, but in which the corresponding substituent is xe2x80x94OPp in place of hydroxy, wherein Pp is a phenol protecting group other than (1-4C)alkyl or benzyl. Phenol protecting groups are well known in the art, for example as described in T. W. Greene and P. G. M. Wuts, xe2x80x9cProtecting Groups in Organic Synthesisxe2x80x9d (1991). Further, Pp may denote a functionalized resin, for example as disclosed in H. V. Meyers, et al., Molecular Diversity, (1995), 1, 13-20.
As mentioned above, the invention includes a pharmaceutically acceptable salt of the factor Xa inhibiting compound defined by the above formula I. A basic compound of this invention possesses one or more functional groups sufficiently basic to react with any of a number of inorganic and organic acids affording a physiologically acceptable counterion to form a pharmaceutically acceptable salt. Acids commonly employed to form pharmaceutically acceptable acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromobenzenesulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of such pharmaceutically acceptable salts thus are the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycollate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like. Preferred pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid, hydrobromic acid and sulfuric acid.
For a compound of formula I which bears an acidic moiety, such as a carboxy group, a pharmaceutically acceptable salt may be made with a base which affords a pharmaceutically acceptable cation, which includes alkali metal salts (especially sodium and potassium), alkaline earth metal salts (especially calcium and magnesium), aluminum salts and ammonium salts, as well as salts made from physiologically acceptable organic bases such as triethylamine, morpholine, piperidine and triethanolamine.
If not commercially available, a necessary starting material for the preparation of a compound of formula I may be prepared by a procedure which is selected from standard techniques of organic chemistry, including aromatic and heteroaromatic substitution and transformation, from techniques which are analogous to the syntheses of known, structurally similar compounds, and techniques which are analogous to the above described procedures or procedures described in the Examples. It will be clear to one skilled in the art that a variety of sequences is available for the preparation of the starting materials. Starting materials which are novel provide another aspect of the invention.
Selective methods of substitution, protection and deprotection are well known in the art for preparation of a compound such as one of formula II, III, IV or VI discussed above.
Generally, a basic compound of the invention is isolated best in the form of an acid addition salt. A salt of a compound of formula I formed with an acid such as one of those mentioned above is useful as a pharmaceutically acceptable salt for administration of the antithrombotic agent and for preparation of a formulation of the agent. Other acid addition salts may be prepared and used in the isolation and purification of the compounds.
As noted above, the optically active isomers and diastereomers of the compounds of formula I are also considered part of this invention. Such optically active isomers may be prepared from their respective optically active precursors by the procedures described above, or by resolving the racemic mixtures. This resolution can be carried out by derivatization with a chiral reagent followed by chromatography or by repeated crystallization. Removal of the chiral auxiliary by standard methods affords substantially optically pure isomers of the compounds of the present invention or their precursors. Further details regarding resolutions can be obtained in Jacques, et al., Enantiomers, Racemates, and Resolutions, John Wiley and Sons, 1981.
The compounds of the invention are believed to selectively inhibit factor Xa over other proteinases and nonenzyme proteins involved in blood coagulation without appreciable interference with the body""s natural clot lysing ability (the compounds have a low inhibitory effect on fibrinolysis). Further, such selectivity is believed to permit use with thrombolytic agents without substantial interference with thrombolysis and fibrinolysis.
The invention in one of its aspects provides a method of inhibiting factor Xa in mammals comprising administering to a mammal in need of treatment an effective (factor Xa inhibiting) dose of a compound of formula I.
In another of its aspects, the invention provides a method of treating a thromboembolic disorder comprising administering to a mammal in need of treatment an effective (thromboembolic disorder therapeutic and/or prophylactic amount) dose of a compound of formula I.
The invention in another of its aspects provides a method of inhibiting coagulation in a mammal comprising administering to a mammal in need of treatment an effective (coagulation inhibiting) dose of a compound of formula I.
The factor Xa inhibition, coagulation inhibition and thromboembolic disorder treatment contemplated by the present method includes both medical therapeutic and/or prophylactic treatment as appropriate.
In a further embodiment, the invention relates to treatment, in a human or animal, of a condition where inhibition of factor Xa is required. The compounds of the invention are expected to be useful in mammals, including man, in treatment or prophylaxis of thrombosis and hypercoagulability in blood and tissues. Disorders in which the compounds have a potential utility are in treatment or prophylaxis of thrombosis and hypercoagulability in blood and tissues. Disorders in which the compounds have a potential utility, in treatment and/or prophylaxis, include venous thrombosis and pulmonary embolism, arterial thrombosis, such as in myocardial ischemia, myocardial infarction, unstable angina, thrombosis-based stroke and peripheral arterial thrombosis. Further, the compounds have expected utility in the treatment or prophylaxis of atherosclerotic disorders (diseases) such as coronary arterial disease, cerebral arterial disease and peripheral arterial disease. Further, the compounds are expected to be useful together with thrombolytics in myocardial infarction. Further, the compounds have expected utility in prophylaxis for reocclusion after thrombolysis, percutaneous transluminal angioplasty (PTCA) and coronary bypass operations. Further, the compounds have expected utility in prevention of rethrombosis after microsurgery. Further, the compounds are expected to be useful in anticoagulant treatment in connection with artificial organs, including joint replacement, and cardiac valves. Further, the compounds have expected utility in anticoagulant treatment in hemodialysis and disseminated intravascular coagulation. A further expected utility is in rinsing of catheters and mechanical devices used in patients in vivo, and as an anticoagulant for preservation of blood, plasma and other blood products in vitro. Still further, the compounds have expected utility in other diseases where blood coagulation could be a fundamental contributing process or a source of secondary pathology, such as cancer, including metastasis, inflammatory diseases, including arthritis, and diabetes. The anti-coagulant compound is administered orally or parenterally, e.g. by intravenous infusion (iv), intramuscular injection (im) or subcutaneously (sc).
The specific dose of a compound administered according to this invention to obtain therapeutic and/or prophylactic effects will, of course, be determined by the particular circumstances surrounding the case, including, for example, the compound administered, the rate of administration, the route of administration, and the condition being treated.
A typical daily dose for each of the above utilities is between about 0.01 mg/kg and about 1000 mg/kg. The dose regimen may vary e.g. for prophylactic use a single daily dose may be administered or multiple doses such as 3 or 5 times daily may be appropriate. In critical care situations a compound of the invention is administered by iv infusion at a rate between about 0.01 mg/kg/h and about 20 mg/kg/h and preferably between about 0.1 mg/kg/h and about 5 mg/kg/h.
The method of this invention also is practiced in conjunction with a clot lysing agent e.g. tissue plasminogen activator (t-PA), modified t-PA, streptokinase or urokinase. In cases when clot formation has occurred and an artery or vein is blocked, either partially or totally, a clot lysing agent is usually employed. A compound of the invention can be administered prior to or along with the lysing agent or subsequent to its use, and preferably further is administered along with aspirin to prevent the reoccurrence of clot formation.
The method of this invention is also practiced in conjunction with a platelet glycoprotein receptor (IIb/IIIa) antagonist, that inhibits platelet aggregation. A compound of the invention can be administered prior to or along with the IIb/IIIa antagonist or subsequent to its use to prevent the occurrence or reoccurrence of clot formation.
The method of this invention is also practiced in conjunction with aspirin. A compound of the invention can be administered prior to or along with aspirin or subsequent to its use to prevent the occurrence or reoccurrence of clot formation. As stated above, preferably a compound of the present invention is administered in conjunction with a clot lysing agent and aspirin.
This invention also provides a pharmaceutical composition for use in the above described therapeutic method. A pharmaceutical composition of the invention comprises an effective factor Xa inhibiting amount of a compound of formula I in association with a pharmaceutically acceptable carrier, excipient or diluent.
The active ingredient in such formulations comprises from 0.1 percent to 99.9 percent by weight of the formulation. By xe2x80x9cpharmaceutically acceptablexe2x80x9d it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
For oral administration the antithrombotic compound is formulated in gelatin capsules or tablets which may contain excipients such as binders, lubricants, disintegration agents and the like. For parenteral administration the antithrombotic is formulated in a pharmaceutically acceptable diluent e.g. physiological saline (0.9 percent), 5 percent dextrose, Ringer""s solution and the like.
The compound of the present invention can be formulated in unit dosage formulations comprising a dose between about 0.1 mg and about 1000 mg. Preferably the compound is in the form of a pharmaceutically acceptable salt such as for example the sulfate salt, acetate salt or a phosphate salt. An example of a unit dosage formulation comprises 5 mg of a compound of the present invention as a pharmaceutically acceptable salt in a 10 mL sterile glass ampoule. Another example of a unit dosage formulation comprises about 10 mg of a compound of the present invention as a pharmaceutically acceptable salt in 20 mL of isotonic saline contained in a sterile ampoule.
The compounds can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal. The compounds of the present invention are preferably formulated prior to administration.
The present pharmaceutical compositions are prepared by known procedures using well known and readily available ingredients. The compositions of this invention may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art. In making the compositions of the present invention, the active ingredient will usually be admixed with a carrier, or diluted by a carrier, or enclosed within a carrier which may be in the form of a capsule, sachet, paper or other container. When the carrier serves as a diluent, it may be a solid, semi-solid or liquid material which acts as a vehicle, excipient or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, (as a solid or in a liquid medium), soft and hard gelatin capsules, suppositories, sterile injectable solutions, sterile packaged powders, and the like.
The following formulation examples are illustrative only and are not intended to limit the scope of the invention in any way. xe2x80x9cActive ingredient,xe2x80x9d of course, means a compound according to formula I or a pharmaceutically acceptable salt or solvate thereof.
Formulation 1: Hard gelatin capsules are prepared using the following ingredients:
Formulation 2: A tablet is prepared using the ingredients below:
The components are blended and compressed to form tablets each weighing 665 mg.
Formulation 3: An aerosol solution is prepared containing the following components:
The active compound is mixed with ethanol and the mixture added to a portion of the propellant 22, cooled to xe2x88x9230xc2x0 C. and transferred to a filling device. The required amount is then fed to a stainless steel container and diluted with the remainder of the propellant. The valve units are then fitted to the container.
Formulation 4: Tablets, each containing 60 mg of active ingredient, are made as follows:
The active ingredient, starch and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly. The aqueous solution containing polyvinylpyrrolidone is mixed with the resultant powder, and the mixture then is passed through a No. 14 mesh U.S. sieve. The granules so produced are dried at 50xc2x0 C. and passed through a No. 18 mesh U.S. Sieve. The sodium carboxymethyl starch, magnesium stearate and talc, previously passed through a No. 60 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 150 mg.
Formulation 5: Capsules, each containing 80 mg of active ingredient, are made as follows:
The active ingredient, cellulose, starch, and magnesium stearate are blended, passed through a No. 45 mesh U.S. sieve, and filled into hard gelatin capsules in 200 mg quantities.
Formulation 6: Suppositories, each containing 225 mg of active ingredient, are made as follows:
The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2 g capacity and allowed to cool.
Formulation 7: Suspensions, each containing 50 mg of active ingredient per 5 mL dose, are made as follows:
The active ingredient is passed through a No. 45 mesh U.S. sieve and mixed with the sodium carboxymethyl cellulose and syrup to form a smooth paste. The benzoic acid solution, flavor and color are diluted with a portion of the water and added, with stirring. Sufficient water is then added to produce the required volume.
Formulation 8: An intravenous formulation may be prepared as follows:
The solution of the above ingredients generally is administered intravenously to a subject at a rate of 1 mL per minute.
The ability of a compound of the present invention to be an effective and orally active factor Xa inhibitor may be evaluated in one or more of the following assays or in other standard assays known to those in the art.
The inhibition by a compound of the inhibition of a serine protease of the human blood coagulation system or of the fibrinolytic system, as well as of trypsin, is determined in vitro for the particular enzyme by measuring its inhibitor binding affinity in an assay in which the enzyme hydrolyzes a particular chromogenic substrate, for example as described in Smith, G. F.; Gifford-Moore, D.; Craft, T. J.; Chirgadze, N.; Ruterbories, K. J.; Lindstrom, T. D.; Satterwhite, J. H. Efegatran: A New Cardiovascular Anticoagulant. New Anticoagulants for the Cardiovascular Patient; Pifarre, R., Ed.; Hanley and Belfus, Inc.: Philadelphia, 1997; pp. 265-300. The inhibitor binding affinity is measured as apparent association constant Kass which is the hypothetical equilibrium constant for the reaction between enzyme and the test inhibitor compound (I).             Enzyme      +      I        ⇄          Enzyme      -      I                  Kass      =                        [                      Enzyme            -            I                    ]                          [                                    (              Enzyme              )                        xc3x97                          (              I              )                                ]                      ⁢          xe2x80x83      
Conveniently, enzyme inhibition kinetics are performed in 96-well polystyrene plates and reaction rates are determined from the rate of hydrolysis of appropriate p-nitroanilide substrates at 405 nm using a Thermomax plate reader from Molecular Devices (San Francisco, Calif.). The same protocol is followed for all enzymes studied: 50 xcexcL buffer (0.03 M Tris, 0.15 M NaCl pH 7) in each well, followed by 25 xcexcL of inhibitor solution (in 100% methanol, or in 50% v:v aqueous methanol) and 25 xcexcL enzyme solution; within two minutes, 150 xcexcL aqueous solution of chromogenic substrate (0.25 mg/mL) is added to start the enzymatic reaction. The rates of chromogenic substrate hydrolysis reactions provide a linear relationship with the enzymes studied such that free enzyme can be quantitated in reaction mixtures. Data is analyzed directly as rates by the Softmax program to produce [free enzyme] calculations for tight-binding Kass determinations. For apparent Kass determinations, 1.34 nM human factor Xa is used to hydrolyze 0.18 mM BzIle-Glu-Gly-Arg-pNA; 5.9 nM human thrombin or 1.4 nM bovine trypsin is used to hydrolyze 0.2 mM BzPhe-Val-Arg-pXA; 3.4 nM human plasmin is used with 0.5 mM HD-Val-Leu-Lys-pNA; 1.2 nM human nt-PA is used with 0.81 mM HD-Ile-Pro-Arg-pNA; and 0.37 nM urokinase is used with 0.30 mM pyro-gfsGlu-Gly-Arg-pNA.
Kass is calculated for a range of concentrations of test compounds and the mean value reported in units of liter per mole. In general, a factor Xa inhibiting compound of formula I of the instant invention, as exemplified herein, exhibits a Kass of 0.1 to 0.5xc3x97106 L/mole or much greater.
The factor Xa inhibitor preferably should spare fibrinolysis induced by urokinase, tissue plasminogen activator (t-PA) and streptokinase. This would be important to the therapeutic use of such an agent as an adjunct to streptokinase, tp-PA or urokinase thrombolytic therapy and to the use of such an agent as an endogenous fibrinolysis-sparing (with respect to t-PA and urokinase) antithrombotic agent. In addition to the lack of interference with the amidase activity of the fibrinolytic proteases, such fibrinolytic system sparing can be studied by the use of human plasma clots and their lysis by the respective fibrinolytic plasminogen activators.
Materials
Dog plasma is obtained from conscious mixed-breed hounds (either sex Butler Farms, Clyde, N.Y., U.S.A.) by venipuncture into 3.8 percent citrate. Fibrinogen is prepared from fresh dog plasma and human fibrinogen is prepared from in-date ACD human blood at the fraction I-2 according to previous procedures and specification. Smith, Biochem. J., 185, 1-11 (1980; and Smith, et al., Biochemistry, 11, 2958-2967, (1972). Human fibrinogen (98 percent pure/plasmin free) is from American Diagnostica, Greenwich, Conn. Radiolabeling of fibrinogen I-2 preparations is performed as previously reported. Smith, et al., Biochemistry, 11, 2958-2967, (1972). Urokinase is purchased from Leo Pharmaceuticals, Denmark, as 2200 Ploug units/vial. Streptokinase is purchased from Hoechst-Roussel Pharmaceuticals, Somerville, N.J.
Methodsxe2x80x94Effects on Lysis of Human Plasma Clots by t-PA
Human plasma clots are formed in micro test tubes by adding 50 xcexcL thrombin (73 NIH unit/mL) to 100 xcexcL human plasma which contains 0.0229 xcexcCi 125-iodine labeled fibrinogen. Clot lysis is studied by overlaying the clots with 50 xcexcL of urokinase or streptokinase (50, 100, or 1000 unit/mL) and incubating for 20 hours at room temperature. After incubation the tubes are centrifuged in a Beckman Microfuge. 25 xcexcL of supernate is added into 1.0 mL volume of 0.03 M tris/0.15 M NaCl buffer for gamma counting. Counting controls 100 percent lysis are obtained by omitting thrombin (and substituting buffer). The factor Xa inhibitors are evaluated for possible interference with fibrinolysis by including the compounds in the overlay solutions at 1, 5, and 10 xcexcg/mL concentrations. Rough approximations of IC50 values are estimated by linear extrapolations from data points to a value which would represent 50 percent of lysis for that particular concentration of fibrinolytic agent.
Anticoagulant Activity
Materials
Dog plasma and rat plasma are obtained from conscious mixed-breed hounds (either sex, Butler Farms, Clyde, N.Y., U.S.A.) or from anesthetized male Sprague-Dawley rats (Harlan Sprague-Dawley, Inc., Indianapolis, Ind., U.S.A.) by venipuncture into 3.8 percent citrate. Fibrinogen is prepared from in-date ACD human blood as the fraction I-2 according to previous procedures and specifications. Smith, Biochem. J., 185, 1-11 (1980); and Smith, et al., Biochemistry, 11, 2958-2967 (1972). Human fibrinogen is also purchased as 98 percent pure/plasmin free from American Diagnostica, Greenwich, Conn. Coagulation reagents Actin, Thromboplastin, Innovin and Human plasma are from Baxter Healthcare Corp., Dade Division, Miami, Fla. Bovine thrombin from Parke-Davis (Detroit, Mich.) is used for coagulation assays in plasma.
Methods
Anticoagulation Determinations
Coagulation assay procedures are as previously described. Smith, et al., Thrombosis Research, 50, 163-174 (1988). A CoAScreener coagulation instrument (American LABor, Inc.) is used for all coagulation assay measurements. The prothrombin time (PT) is measured by adding 0.05 mL saline and 0.05 mL Thromboplastin-C reagent or recombinant human tissue factor reagent (Innovin) to 0.05 mL test plasma. The activated partial thromboplastin time (APTT) is measured by incubation of 0.05 mL test plasma with 0.05 mL Actin reagent for 120 seconds followed by 0.05 mL CaCl2 (0.02 M.). The thrombin time (TT) is measured by adding 0.05 mL saline and 0.05 mL thrombin (10 NIH units/mL) to 0.05 mL test plasma. The compounds of formula I are added to human or animal plasma over a wide range of concentrations to determine prolongation effects on the APTT, PT, and TT assays. Linear extrapolations are performed to estimate the concentrations required to double the clotting time for each assay.
Animals
Male Sprague Dawley rats (350-425 gm, Harlan Sprague Dawley Inc., Indianapolis, Ind.) are anesthetized with xylazine (20 mg/kg, s.c.) and ketamine (120 mg/kg, s.c.) and maintained on a heated water blanket (37xc2x0 C.). The jugular vein(s) is cannulated to allow for infusions.
Arterio-Venous Shunt Model
The left jugular vein and right carotid artery are cannulated with 20 cm lengths of polyethylene PE 60 tubing. A 6 cm center section of larger tubing (PE 190) with a cotton thread (5 cm) in the lumen, is friction fitted between the longer sections to complete the arterio-venous shunt circuit. Blood is circulated through the shunt for 15 min before the thread is carefully removed and weighed. The weight of a wet thread is subtracted from the total weight of the thread and thrombus (see J. R. Smith, Br J Pharmacol, 77:29, 1982).
FeCl3 Model of Arterial Injury
The carotid arteries are isolated via a midline ventral cervical incision. A thermocouple is placed under each artery and vessel temperature is recorded continuously on a strip chart recorder. A cuff of tubing (0.058 IDxc3x970.077 ODxc3x974 mm, Baxter Med. Grade Silicone), cut longitudinally, is placed around each carotid directly above the thermocouple. FeCl3 hexahydrate is dissolved in water and the concentration (20 percent) is expressed in terms of the actual weight of FeCl3 only. To injure the artery and induce thrombosis, 2.85 xcexcL is pipetted into the cuff to bathe the artery above the thermocouple probe. Arterial occlusion is indicated by a rapid drop in temperature. The time to occlusion is reported in minutes and represents the elapsed time between application of FeCl3 and the rapid drop in vessel temperature (see K. D. Rurz, Thromb. Res., 60:269, 1990).
Coagulation Parameters
Plasma thrombin time (TT) and activated partial thromboplastin time (APTT) are measured with a fibrometer. Blood is sampled from a jugular catheter and collected in syringe containing sodium citrate (3.8 percent, 1 part to 9 parts blood). To measure TT, rat plasma (0.1 mL) is mixed with saline (0.1 mL) and bovine thrombin (0.1 mL, 30 U/mL in TRIS buffer; Parke Davis) at 37xc2x0 C. For APTT, plasma (0.1 mL) and APTT solution (0.1 mL, Organon Teknika) are incubated for 5 minutes (37xc2x0 C.) and CaCl2 (0.1 mL, 0.025 M) is added to start coagulation. Assays are done in duplicate and averaged.
Index of Bioavailability
Bioavailability studies may be conducted as follows. Compounds are administered as aqueous solutions to male Fisher rats, intravenously (iv) at 5 mg/kg via tail vein injection and orally (po) to fasted animals at 20 mg/kg by gavage. Serial blood samples are obtained at 5, 30, 120, and 240 minutes postdose following intravenous administration and at 1, 2, 4, and 6 hours after oral dosing. Plasma is analyzed for drug concentration using an HPLC procedure involving C8 Bond Elute (Varion) cartridges for sample preparation and a methanol/30 nM ammonium acetate buffer (pH 4) gradient optimized for each compound. % Oral bioavailability is calculated by the following equation:       %    ⁢          xe2x80x83        ⁢    Oral    ⁢          xe2x80x83        ⁢    bioavailability    =                    AUC        ⁢                  xe2x80x83                ⁢        po                    AUC        ⁢                  xe2x80x83                ⁢        iv              xc3x97                  Dose        ⁢                  xe2x80x83                ⁢        iv                    Dose        ⁢                  xe2x80x83                ⁢        po              xc3x97    100  
where AUC is area under the curve calculated from the plasma level of compound over the time course of the experiment following oral (AUC po) and intravenous (AUC iv) dosing.
Compounds
Compound solutions are prepared fresh daily in normal saline and are injected as a bolus or are infused starting 15 minutes before and continuing throughout the experimental perturbation which is 15 minutes in the arteriovenous shunt model and 60 minutes in the FeCl3 model of arterial injury and in the spontaneous thrombolysis model. Bolus injection volume is 1 mL/kg for i.v., and 5 mL/kg for p.o., and infusion volume is 3 mL/hr.
Statistics
Results are expressed as means+/xe2x88x92SEM. One-way analysis of variance is used to detect statistically significant differences and then Dunnett""s test is applied to determine which means are different. Significance level for rejection of the null hypothesis of equal means is P less than 0.05.
Animals
Male dogs (Beagles; 18 months-2 years; 12-13 kg, Marshall Farms, North Rose, N.Y. 14516) are fasted overnight and fed Purina certified Prescription Diet (Purina Mills, St. Louis, Mo.) 240 minutes after dosing. Water is available ad libitum. The room temperature is maintained between 66-74xc2x0 F.; 45-50 percent relative humidity; and lighted from 0600-1800 hours.
Pharmacokinetic Model
Test compound is formulated immediately prior to dosing by dissolving in sterile 0.9 percent saline to a 5 mg/mL preparation. Dogs are given a single 2 mg/kg dose of test compound by oral gavage. Blood samples (4.5 mL) are taken from the cephalic vein at 0.25, 0.5, 0.75, 1, 2, 3, 4 and 6 hours after dosing. Samples are collected in citrated Vacutainer tubes and kept on ice prior to reduction to plasma by centrifugation. Plasma samples are analyzed by HPLC MS. Plasma concentration of test compound is recorded and used to calculate the pharmacokinetic parameters: elimination rate constant, Ke; total clearance, Clt; volume of distribution, VD; time of maximum plasma test compound concentration, Tmax; maximum concentration of test compound of Tmax, Cmax; plasma half-life, t0.5; and area under the curve, A.U.C.; fraction of test compound absorbed, F.
Canine Model of Coronary Artery Thrombosis
Surgical preparation and instrumentation of the dogs are as described in Jackson, et al., Circulation, 82, 930-940 (1990). Mixed-breed hounds (aged 6-7 months, either sex, Butler Farms, Clyde, N.Y., U.S.A.) are anesthetized with sodium pentobarbital (30 mg/kg intravenously, i.v.), intubated, and ventilated with room air. Tidal volume and respiratory rates are adjusted to maintain blood PO2, PCO2, and pH within normal limits. Subdermal needle electrodes are inserted for the recording of a lead II ECG.
The left jugular vein and common carotid artery are isolated through a left mediolateral neck incision. Arterial blood pressure (ABP) is measured continuously with a precalibrated Millar transducer (model (MPC-500, Millar Instruments, Houston, Tex., U.S.A.) inserted into the carotid artery. The jugular vein is cannulated for blood sampling during the experiment. In addition, the femoral veins of both hindlegs are cannulated for administration of test compound.
A left thoracotomy is performed at the fifth intercostal space, and the heart is suspended in a pericardial cradle. A 1- to 2-cm segment of the left circumflex coronary artery (LCX) is isolated proximal to the first major diagonal ventricular branch. A 26-gauge needle-tipped wire anodal electrode (Teflon-coated, 30-gauge silverplated copper wire) 3-4 mm long is inserted into the LCX and placed in contact with the intimal surface of the artery (confirmed at the end of the experiment). The stimulating circuit is completed by placing the cathode in a subcutaneous (s.c.) site. An adjustable plastic occluder is placed around the LCX, over the region of the electrode. A precalibrated electromagnetic flow probe (Carolina Medical Electronics, King, N.C., U.S.A.) is placed around the LCX proximal to the anode for measurement of coronary blood flow (CBF). The occluder is adjusted to produce a 40-50 percent inhibition of the hyperemic blood flow response observed after 10-s mechanical occlusion of the LCX. All hemodynamic and ECG measurements are recorded and analyzed with a data acquisition system (model M3000, Modular Instruments, Malvern, Pa. U.S.A.).
Thrombus Formation and Compound Administration Regimens
Electrolytic injury of the intima of the LCX is produced by applying 100-xcexcA direct current (DC) to the anode. The current is maintained for 60 min and then discontinued whether the vessel has occluded or not. Thrombus formation proceeds spontaneously until the LCX is totally occluded (determined as zero CBF and an increase in the S-T segment). Compound administration is started after the occluding thrombus is allowed to age for 1 hour. A 2-hour infusion of the compounds of the present invention at doses of 0.5 and 1 mg/kg/hour is begun simultaneously with an infusion of thrombolytic agent (e.g. tissue plasminogen activator, streptokinase, APSAC). Reperfusion is followed for 3 hour after administration of test compound. Reocclusion of coronary arteries after successful thrombolysis is defined as zero CBF which persisted for at least 30 minutes.
Hematology and Template Bleeding Time Determinations
Whole blood cell counts, hemoglobin, and hematocrit values are determined on a 40-xcexcL sample of citrated (3.8 percent) blood (1 part citrate:9 parts blood) with a hematology analyzer (Cell-Dyn 900, Sequoia-Turner. Mount View, Calif., U.S.A.). Gingival template bleeding times are determined with a Simplate II bleeding time device (Organon Teknika Durham, N.C., U.S.A.). The device is used to make 2 horizontal incisions in the gingiva of either the upper or lower left jaw of the dog. Each incision is 3 mm widexc3x972 mm deep. The incisions are made, and a stopwatch is used to determine how long bleeding occurs. A cotton swab is used to soak up the blood as it oozes from the incision. Template bleeding time is the time from incision to stoppage of bleeding. Bleeding times are taken just before administration of test compound (0 min), 60 min into infusion, at conclusion of administration of the test compound (120 min), and at the end of the experiment.
All data are analyzed by one-way analysis of variance (ANOVA) followed by Student-Neuman-Kuels post hoc t test to determine the level of significance. Repeated-measures ANOVA are used to determine significant differences between time points during the experiments. Values are determined to be statistically different at least at the level of p less than 0.05. All values are meanxc2x1SEM. All studies are conducted in accordance with the guiding principles of the American Physiological Society. Further details regarding the procedures are described in Jackson, et al., J. Cardiovasc. Pharmacol., (1993), 21, 587-599.
The following Examples are provided to further describe the invention and are not to be construed as limitations thereof.
The abbreviations, symbols and terms used in the examples have the following meanings.
Ac=acetyl
aq=aqueous
Bn or Bzl=benzyl
Boc=t-butyloxycarbonyl
Bu=butyl
n-BuLi=butyllithium
Calcd=calculated
conc=concentrated
DMF=dimethylformamide
DMSO=dimethylsulfoxide
eq=(molar) equivalent
Et=ethyl
EtOAc=ethyl acetate
Et3N=triethylamine
Et2O=diethyl ether
EtOH=ethanol
FTIR=Fourier transform IR
Hex=hexanes
HPLC=High Performance Liquid Chromatography
HRMS=high resolution mass spectrum
i-PrOH=isopropanol
IR=Infrared Spectrum
LC-MS=liquid chromatographyxe2x80x94mass spectrum (using HPLC)
Me=methyl
MeOH=methanol
MS-ES (or ES-MS)=electrospray mass spectrum
MS-FAB (or FAB-MS)=fast atom bombardment mass spectrum
MS-FIA (or FIA-MS)=flow injection analysis mass spectrum
MS-FD (or FD-MS)=field desorption mass spectrum
MS-IS (or IS-MS)=ion spray mass spectrum
NMR=Nuclear Magnetic Resonance
Ph=phenyl
i-Pr=isopropyl
RPHPLC=Reversed Phase High Performance Liquid Chromatography
RT (or Rt)=retention time
satd=saturated
SiO2=silica gel
SCX=strong cation exchange (resin)
TBS=tert-butyldimethylsilyl
TFA=trifluoroacetic acid
THF=tetrahydrofuran
TIPS=triisopropylsilyl
TLC=thin layer chromatography
tosyl=p-toluenesulfonyl
triflic acid=trifluoromethanesulfonic acid
Unless otherwise stated, pH adjustments and work up are with aqueous acid or base solutions. 1H-NMR indicates a satisfactory NMR spectrum was obtained for the compound described. IR (or FTIR) indicates a satisfactory infra red spectrum was obtained for the compound described.
For consistency and clarity, a number of compounds are named as substituted benzamide derivatives.
Analytical HPLC method was a linear gradient of 90/10 to 50/50 (0.1% TFA in water/0.1% TFA in acetonitrile) over 40 minutes with a flow rate of 1 mL/min.
LC-MS Method A: gradient from 50% acetonitrile-40% water-10% water with 0.1% trifluoroacetic acid to 90% acetonitrile-10% water with 0.1% trifluoroacetic acid over 5 min; hold 5 min; 0.5 mL/min; Zorbax SB-C18 column, 4.6 by 75 mm; 25xc2x0 C. LC-MS Method B: gradient from 20% acetonitrile-70% water-10% water with 0.1% trifluoroacetic acid to 70% acetonitrile-20% water-10% water with 0.1% trifluoroacetic acid over 5 min; hold 5 min; 0.5 mL/min; Zorbax SB-C18 column, 4.6 by 75 mm; 25xc2x0 C.